Cells have multiple compartments, each with a unique set of proteins which support specialized functions. These compartments are separated by lipid-rich membranes. How does a cell sort newly made proteins to their correct compartments and transport them across the appropriate membranes? To address this question, mutations in the export pathway of E. coli have been isolated. Cells bearing these mutations exhibit a conditionally lethal phenotype. E. coli mutants in pr1A (secY) show defective export of proteins to the periplasm and outer membrane. Recently we have shown that pr1A (secY) can function in a posttranslational manner and that pr1A (secY) is required for translocation of proOmpA into inner membrane vesicles in vitro. This finding will allow a complementation assay to be developed for isolation of a functional pr1A (secY) protein. Studies examining the membrane topology of pr1A (secY) and its binding to leader peptides and preproteins are proposed. Other functional characterizations will include the interaction of pr1A (secY) with ATP, its influence on the generation and maintenance of the membrane electrochemical potential, and its possible interaction with soluble components of the export machinery such as secA. The eventual goal of this research is to purify the pr1A (secY) protein in a functional state and determine its catalytic role in protein export.